Protein kinases are of particular interest in drug discovery research because they have been shown to be key regulators of many cell functions, including signal transduction (Ullrich and Schlessinger, 1990), transcriptional regulation (Pawson and Bernstein, 1990), cell motility (Miglietta and Nelson, 1988) and cell division (Pines and Hunter, 1990). Protein kinases are enzymes which covalently modify proteins and peptides by the attachment of a phosphate group to one or more sites on the protein. Phosphatases perform the opposite function. Many of the known protein kinases use adenosine triphosphate (ATP) as the phosphate donor, placing the γ-phosphate onto a histidine, tyrosine, serine or threonine residue in the protein. The location of the modification site and the type of residue modified by the kinase are usually specific for each particular kinase.
The added phosphate alters certain structural, thermodynamic and kinetic properties of the phosphorylated protein. Generally, the phosphate adds two negative charges to the protein. This modifies the electrostatic interactions between the protein's constituent amino acids, in turn altering secondary and tertiary protein structure. The phosphate may also form up to three hydrogen bonds or salt bridges with other protein residues, or may otherwise change the conformational equilibrium between different functional states of the protein. These structural changes provide the basis, in a biological system, for altering substrate binding and catalytic activity of the phosphorylated proteins.
Phosphorylation and dephosphorylation reactions, under the control of kinases and phosphatases, respectively, can occur rapidly to form stable structures. This makes the phosphorylation system ideal as a regulatory process. Phosphorylation and dephosphorylation reactions may also be part of a cascade of reactions that can amplify a signal that has an extracellular origin, such as hormones and growth factors.
Methods for assaying the activity of protein kinases often utilize a synthetic peptide substrate that can be phosphorylated by the kinase protein under study. The most common mechanisms for detecting phosphorylation of the peptide substrates are 1) Incorporation of 32P (or 33P) phosphate from [32P]γ-ATP into the peptides, purification of the peptides from ATP, and scintillation or Cherenkov counting of the incorporated radionucleotide, 2) Detection of phosphoamino acids with radiolabeled specific antibodies, or 3) Purification of phosphorylated peptides from unphosphorylated peptides by chromatographic or electrophoretic methods, followed by quantification of the purified product.
For example, in one widely used method, a sample containing the kinase of interest is incubated with activators and a substrate in the presence of gamma 32P-ATP, with an inexpensive substrate, such as histone or casein being used. After a suitable incubation period, the reaction is stopped and an aliquot of the reaction mixture is placed directly onto a filter that binds the substrate. The filter is then washed several times to remove excess radioactivity, and the amount of radiolabelled phosphate incorporated into the substrate is measured by scintillation counting (Roskoski, 1983).
The use of 32P in assays, however, poses significant disadvantages. One major problem is that, for sensitive detection, relatively high quantities of 32P must be used routinely and subsequently disposed. The amount of liquid generated from the washings is not small, and contains 32P. Due to government restrictions, this waste cannot be disposed of easily. It must be allowed to decay, usually for at least six months, before disposal. Another disadvantage is the hazard posed to personnel working with the isotope. Shielding and special waste containers are inconvenient but necessary for safe handling of the isotope. Further, the lower detection limit of the assay is determined by the level of background phosphorylation and is therefore variable. In short, the study of protein kinases would be greatly facilitated by the development of an efficient and accurate assay that does not require the use of radioactivity.
Although radioisotope methods have been applied in high throughput screening, the high cost and strict safety regulation incurred with the use of radioisotopes in high throughput screening greatly limits their use in drug discovery. For these and other reasons, it would be useful to develop alternative methods and apparatus for high throughput screening that facilitate measuring the kinase dependent phosphorylation of peptides.
Recently, several analytical chemistry research groups have experimented with micro-lithographed electrophoretic separation devices. These devices typically contain four or more reservoirs connected by a cross-shaped arrangement of channels. A long, sinuous channel is usually situated at the tail of the cross, which is utilized for electrophoretic separation of charged molecules in the system. These devices rely on electro osmotic flow (EOF) forces to provide flow of the solution through the system, and thus all molecules (positive, negative, and uncharged) in the sample are transported in the same direction along the separation channel, their speed and position determined by their net charge/mass. In order to detect each molecule in the sample, a continuous detection system is used during the electrophoretic separation process. Because of their reliance on EOF forces, these devices must be manufactured to high tolerance (with small cross-section microchannels) and are designed to include a rather long separation path.